Redox reactions involving cysteine thiol-disulfide exchange are crucial for sensing intracellular levels of H(2)O(2). However, oxidation-sensitive dithiols are also sensitive to intracellular reducing agents, and disulfide bonds are thus transient. The yeast transcription factor Yap1 is activated by disulfide-induced structural changes in the nuclear export signal in a carboxy-terminal domain. We show herein that the activation of Yap1 by H(2)O(2) requires multistep formation of disulfide bonds. One disulfide bond forms within 15 s in an amino-terminal domain, and then disulfide bonds linking the two domains accumulate. The multiple interdomain disulfide bonds, which result in reduction-resistant Yap1, are required for transduction of the H(2)O(2) stress signal to induce the appropriate level and duration of specific transcription. Our results suggest both a mechanism wherein the H(2)O(2) levels might be sensed by Yap1 and the way in which the NADPH levels might be maintained by altering the redox status of Yap1.

CorrectionsBIOCHEMISTRY. For the article ''Interaction of RNA polymerase with forked DNA: Evidence for two kinetically significant intermediates on the pathway to the final complex,'' by Laura Tsujikawa, Oleg V. Tsodikov, and Pieter L. deHaseth, which appeared in number 6, March 19, 2002, of Proc. Natl. Acad. Sci. USA (99, 3493-3498; First Published March 12, 2002; 10.1073͞ pnas.062487299), the authors note the following concerning RNA polymerase (RNAP) concentrations. No correction was made for the fraction of RNAP (0.5) that is active in promoter binding. With this correction, the values of K 1 and K app (but not K f ) would increase by about a factor of 2. The relative values would remain essentially unchanged. Also, the legends to Figs. 2, 3, and 5 contain errors pertaining to the symbols used for data obtained with and without heparin challenge, the duration of the challenge, and the concentration of added heparin. The figures and the corrected legends appear below. Fig. 2. Determination of equilibrium affinities by titration of wt Fork with RNAP. The reactions contained 1 nM wt Fork and variable amounts of RNAP as shown and were analyzed by electrophoretic mobility shift immediately (OE; data shown are averages of three independent experiments) or after a challenge with 100 g͞ml heparin for 10 min (F; data shown are averages of four independent experiments). The curves shown reflect the simultaneous errorweighted fits of the data to Eqs. 3 and 4 -7. The parameters are shown in Table 1 (line 1).www.pnas.org͞cgi͞doi͞10.1073͞pnas.013667699 Fig. 3. Kinetics of complex formation. RNAP (65 nM) and wt forked DNA (1 nM) were incubated for various time intervals and then complex formation was determined immediately (Ϫheparin) or after a 2-min challenge with 100 g͞ml heparin (ϩheparin). The Ϫheparin data (s) were fit (error-weighted) with Eq. 8 with a 2 ϭ 0 (kaϪ ϭ 0.10 Ϯ 0.01 s Ϫ1 ) and the ϩheparin data (OE) with both single (k aϩ ϭ 0.036 Ϯ 0.004 s Ϫ1 ; thin line) and double-exponential (ka 1 ϭ 0.044 Ϯ 0.002 s Ϫ1 ; ka 2 ϭ (5 Ϯ 3) ϫ 10 Ϫ4 s Ϫ1 ; thick line) equations. Fig. 5.Comparison of the kinetics for formation and dissociation of competitor-resistant complexes between RNAP and wt Fork. Association data were obtained as described in the text and the legend for Fig. 3 except the concentration of forked DNA was 10 nM. Dissociation kinetics were obtained by challenging with 100 g͞ml heparin a mixture of RNAP and forked DNA that had been incubated for 30 min. The curves represent double-exponential fits of the data to Eq. 10. (A) wt RNAP. The observed association rate constants (s) are shown in the legend for Fig. 3; for the slow phase of the dissociation of the wt Fork-wt RNAP complex (F), kd 2 ϭ (1.3 Ϯ 0.2) ϫ 10 Ϫ4 s Ϫ1 . (B) YYW RNAP. The slow phase of the association reaction (F) has a ka 2 ϭ (1.1 Ϯ 0.3) ϫ 10 Ϫ3 s Ϫ1 ; the slow phase of the dissociation reaction (s), a kd 2 ϭ (6 Ϯ 1) ϫ 10 Ϫ4 s Ϫ1 . 4 should have appeared in color. In addition, the marker for the Ub band of Fig. 6A should be 14. The corrected...

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A redox reaction involving cysteine thiol-disulfide exchange is crucial for the intracellular monitoring of oxidation status. The yeast transcription factor Yap1 is activated by formation of a disulfide bond, which inhibits nuclear export in response to peroxide stress, with resultant enhancement of the nuclear localization of Yap1. A glutathione peroxidase-like protein, Gpx3, which has peroxiredoxin activity, is required for formation of the disulfide bond in Yap1. We show here that the requirement for Gpx3 in the regulation of Yap1 is strain-specific. Thus, Tsa1, a ubiquitous thioredoxin peroxidase, is required for the activation of Yap1 in yeast strain Y700, which is derived from W303. The strain-specific utilization of different peroxiredoxins appears to be determined by Ybp1, a Yap1-binding protein. The Ybp1 of Y700 has a nonsense mutation, and a wild-type YBP1 gene can restore the Gpx3-dependent activation of Yap1. These results suggest that Tsa1, a ubiquitous peroxiredoxin, has the potential for transducing redox signals to a particular sensor protein.